Proteinaceous molecules, such as enzymes, hormones, storage proteins, binding proteins and transport proteins may be produced by recombinant DNA techniques. For instance, DNA fragments coding for selected proteins, together with promoter sequences are ligated to an appropriate vector in the presence of ligating enzymes. The recombinant vector is inserted within host prokaryotic or eukaryotic cells. Transformed host cells are identified, isolated and then cultivated to achieve multiple copies (replication) of the recombinant DNA vector and/or expression of the protein or polypeptides coded for by the foreign DNA.
To enable the foregoing procedures to be used as a viable method of producing proteinaceous products on a commercial scale, intense efforts are being made to increase the efficiency of such procedures to achieve higher output levels of recombinant DNA products. One possible technique for increasing the yield of such products is by identifying and employing more highly efficient promoters. As used herein, the term "promoter" refers to a DNA segment capable of functioning to initiate transcription of an adjoining DNA segment. Transcription is the synthesis of messenger RNA ("mRNA") complementary to one strand of the DNA adjoining the promoter region. In prokaryotic host cells, mRNA synthesis is catalyzed by RNA polymerase, which is the same enzyme employed in ribosomal RNA ("rRNA") synthesis and transfer RNA ("tRNA") synthesis. In eukaryotic cells, the synthesis of the three forms of RNA are catalyzed by distinct polymerases with eukaryotic mRNA syntheses catalyzed by RNA polymerase II.
The promoter provides the binding site for RNA polymerase or RNA polymerase II so that the proper strand of DNA serves as a template for mRNA synthesis. The promoter also regulates the rate at which transcription occurs. Some promoters are more accurate than others and/or regulate the synthesis of much larger quantities of mRNA than produced by less active promoters. As such, protein expression is enhanced by selecting efficient promoters compatible with the host cells. An example of an efficient promoter for plasmid vectors effective in Escherichia coli ("E. coli") host cells is the promoter from the lac operon. Efficient promoters for protein expression in yeast cells include the promoters for alcohol dehydrogenase ("ADH") or the alpha-factor gene, Kurjan and Herskowitz (1982) Cell, 30:933-943. Examples of the use of promoters in yeast vectors from the gene coding for phosphoglyceride kinase are set forth in U.S. Pat. No. 4,615,974.
For the cloning of genes and the production of desired products in eukaryotic hosts, viral vectors are commonly employed, for instance from the simian virus 40 ("SV40"), polyoma, human BK virus ("BK") and adeno viruses. These virions include strong promoters. The viral vectors are used in their "natural" form and also in mutated form. Moreover, not uncommonly, the promoter region of the viral DNA is utilized in nonviral derived vectors, such as vectors originating from the pBR322 E. coli bacterial plasmid. Examples of nonviral derived vectors utilizing viral promoter regions are disclosed in U.S. Pat. Nos. 4,510,245 and 4,562,155.
In addition to employing promoters in their "natural" state, attempts have been made to increase vector replication and expression of protein products by development of mutated promoters. An example of such a promoter is disclosed in U.S. Pat. No. 4,374,927.
In addition to promoters, transcription of DNA in viral hosts is facilitated by enhancers. Enhancer sequences have been found in the DNA of several viruses of higher eukaryotes, including the SV40, virus murine leukemia virus, polyoma virus, bovine papilloma virus and BK virus. The enhancer sequences typically greatly increase the transcription of the gene from virtually any nearby promoter. They may be located great distances upstream or downstream from the gene. It is not known how the enhancer sequences exert their effect. One theory is that enhancer sequences maintain the DNA in an open, protein-free confirmation, thus providing entry sites for RNA polymerase. Except for a "consensus sequence" of from about seven to ten base pairs that have been found in a number of enhancers, there seems to be little homology among the various viral enhancers which have been identified.
The use of enhancers to increase transcription of DNA has been described in the literature. For example, Rosenthal et al. (1983) Science, 222: 749-754, describes the use of the BK viral enhancer to increase transcription. Also, the enhancer sequences from SV40 are commonly employed in cloning and expression vectors. See, for instance, Old and Primrose, Principles of Gene Manipulation, 2nd Ed. pp. 121 et seq., 1981.